Project Summary With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. In particular, next generation therapeutics derived from monoclonal antibodies (e.g. Fab, scAb, scFv, immunotoxins, etc) show great clinical promise in treating a range of human disorders including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Despite the tremendous progress to date, the use of antibodies and antibody derivatives for cancer treatment remains limited, leaving great potential for further improvements. One type of antibody derivative that has shown great promise for cancer treatment is bispecific antibodies (BsAbs), which redirect effector cells towards therapeutic targets by virtue of their two distinct specificities. Numerous methods for constructing BsAbs have emerged including hybrid hybridomas, chemical crosslinking, renaturation from bacterial inclusion bodies, and the use of non-covalent coupling in diabodies or single-chain antibody fragments (scFvs) with multimerization domains/tags, yet each of these suffers from complexity of production, low yields, ill-defined by-products, laborious purification procedures and high cost of goods. One strategy that overcomes these deficiencies is to covalently couple two scFvs by a flexible polypeptide linker on a single polypeptide chain. These chimeras, known as single- chain bispecific antibodies or tandem scFvs, can be successfully expressed in mammalian cells. Unfortunately, their functional expression in a more robust, cost-effective system such as E. coli has been elusive. Hence, these studies seek to develop a novel technology platform for the creation of functional bispecific scFvs in E. coli with the potential to solve the production issues (e.g., complexity of BsAb molecules, low yields, high costs) that have plagued the field of BsAbs since its inception more than 20 years ago. Moreover, an E. coli expression platform would enable molecular engineering (e.g., affinity maturation via directed evolution) of tandem scFvs that is currently intractable with most existing BsAb expression systems. To achieve this goal, the following specific aims are proposed: (1) construct and engineer single-chain bispecific antibodies that are stably expressed in E. coli;and (2) validate the bacterial Tat protein export system for genetic selection of dual antigen binding. We anticipate that the successful completion of these aims will result in a technology platform with the potential to greatly decrease the time and cost associated with discovery and development of functional tandem scFvs for pre-clinical and clinical investigation. PUBLIC HEALTH RELEVANCE: Project Narrative Bispecific antibodies (BsAbs) comprised of two specificities can effectively redirect effector cells towards therapeutic targets and thus hold great promise for cancer treatment. However, the therapeutic potential of BsAbs has not been fully realized due to inefficient production methods, especially in Escherichia coli where several different BsAb formats have been met with limited success. Hence, the proposed studies seek to exploit the remarkable features of a natural bacterial protein export mechanism to create an all-in-one technology platform for the expression and engineering of functional bispecific single-chain Fv antibodies in E. coli.